Kalkitoxin, a toxin derived from the cyanobacterium Lyngbya majuscula, induces NMDA receptor mediated neuronal necrosis, blocks voltage-dependent sodium channels, and induces cellular hypoxia by inhibiting the electron transport chain (ETC) complex 1.
Natural sources Kalkitoxin is an ichthyotoxin, derived from the cyanobacterium Lyngbya majuscula which covers sections of the coral reef. It typically forms mini-blooms and produces several metabolites, such as kalkitoxin, curacin-A and antillatoxin. Kalkitoxin has been found and purified near the coasts of Curaçao and Puerto Rico.
Structure and reactivity Kalkitoxin is a lipopeptide toxin with a molecular weight of 366.604Da. Its chemical formula is C21H38N2OS. The structure contains two double bonds, a 2,4-disubstituted thiazoline ring system, and an additional carbonyl-group. These four groups each provide a degree of unsaturation, which causes kalkitoxin to have four degrees of unsaturation. The structure contains 5 chiral centers, one of which is due to a substituent of the thiazoline ring, and the other four are due to methine groups along the aliphatic carbon chain, which are tertiary carbon atoms bearing three single carbon bonds and one hydrogen. The four methyl groups (each at a methine chiral center), the structure's overall stereochemistry, and the N-methyl group all contribute to the toxicity of kalkitoxin.
Structure determination The structure of kalkitoxin was first determined by characterizing six partial structures which were subsequently connected to yield the total structure. This investigation was largely carried out through various NMR experiments. Structure (a) is a sec-butyl group, indicated by characteristic deshielding of its central methine group due to the adjacent carbonyl. Structure (b) contains this carbonyl group, and an adjacent tertiary methylated nitrogen atom, constituting a tertiary amide group. Since this is a tertiary amide, it exists in a cis/trans mixture, which underlies the two conformations of kalkitoxin. Structure (c) is a string of two methylene groups, then a methine group bearing a high-field methyl group. The next two groups identified (d,e) are identical and opposing strings of CH2-CH-CH3, however the left grouping's methylene protons experience greater deshielding, due to their proximity to the adjacent imine. Deshielding is an effect of a nearby electronegative atom withdrawing electron density from a given atom nucleus, eliciting an increased chemical shift as measured by NMR. The final partial structure consists of a thiazoline ring with a terminal alkene substituent, as determined by electron ionization mass spectrometry (EI-MS) and 13C NMR. The chemical shifts of ring carbons adjacent to the sulfur and nitrogen heteroatoms were compared to 13C NMR data from model compounds. This allowed for the determination of these heteroatoms' locations in the ring, and subsequently the existence of the thiazoline ring itself. With these partial structures established, their connectivity was evaluated via HMBC spectroscopy, a 2D NMR technique which allows for the determination of heteronuclear J-coupling values for nonadjacent carbons and protons. This allows for the spatial relation of specific carbon and hydrogen atoms within a structure to be determined.
Stereochemistry Kalkitoxin has five chiral centers, one of which is the ring carbon to which the terminal alkene is coordinated, with the remaining four occurring at tertiary carbon atoms along the aliphatic chain originating from the imine nitrogen. The total stereochemistry of natural (+)-kalkitoxin is 3R,7R,8S,10S,2′R. For this determination, 3JCH values by a variation of the HSQMBC pulse technique, a type of HMBC spectroscopy, and 3JHH values by exclusive correlation spectroscopy (E.COSY). These methods use NMR to evaluate the spin-spin coupling constants which directly relate to the dihedral angle of the atoms being analyzed, allowing for the determination of chirality. This was used to determine the stereochemistry of chiral centers at C7, C8, and C10. Because C7 and C8 are adjacent stereocenters, these techniques allowed for immediate determination of their relative stereochemistry, however C10 is separated from C8 by C9, which carries two diastereotopic protons. This allows for the determination of relative stereochemistry of C8 and C10 to the C9 protons through 3J coupling values, so as to relate the relative stereochemistry of C8 to C10. These methods yielded a relative stereochemistry of 7R, 8S, 10S for the aliphatic chain stereocenters. Stereochemistry at C3 was determined by Marfey's analysis, wherein the compound was ozonized and subsequently hydrolyzed to obtain cysteic acid from the thiazoline ring and attached terminal alkene. Marfey's analysis indicated this amino acid derivative was L-cysteic acid, indicating R absolute stereochemistry at C3. The absolute stereochemistry of the total molecule was determined by synthesizing the possible configurations of the already determined relative chiralities, and comparison of these to natural Kalkitoxin via 13C NMR shift differences, revealing the natural (+)-kalkitoxin stereochemistry to be 3R,7R,8S,10S,2′R.
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